UC San Diego

All cell types express heparan sulfate proteoglycans (HSPGs) where they are embedded into the cell membrane or released into the extracellular matrix. To identify novel membrane bound heparan sulfate binding proteins (HSBPs), we utilized limited proteolysis to liberate ectodomains of cell surface proteins expressed on human umbilical vein endothelial cells. Chromatography over heparin-Sepharose and mass spectrometry yielded several known HSBPs while also isolating the heparin binding domain. Novel endothelial HSPBs were identified including PTPRB, CLEC14A, CLEC2B, CD93, and GDF15. We mapped the heparin-binding domain of CLEC14A by mutagenesis and showed that the binding site resides in the C-type lectin domain. Recombinant CLEC14A ectodomain bound with high affinity heparin oligosaccharides of ≥dp6. Binding occurred in 1:1 stoichiometry and led to increased thermal stability of CLEC14A. Overexpression of membrane bound wild-type CLEC14A or the ectodomain had no effect on in vitro angiogenic sprouting, whereas the CLEC14A heparin binding deficient mutant protein inhibited sprouting. To determine how heparin binding mediates CLEC14A function, we then used chemical cross-linkers to identify CLEC14A endothelial protein binding partners and to test if heparin can alter CLEC14A protein-protein interactions. Mass spectrometry identified over 15 CLEC14A binding partners including MMRN2, ITIH5, NRP1, TXLNA, and EFEMP1. Heparin blocked binding of CLEC14A to MMRN2, but mutation of a key residue in the heparin-binding domain did not interfere with binding. A model is proposed in which heparan sulfate modulates CLEC14A-protein interactions resulting in altered angiogenesis.